IL-17 mediates articular hypernociception in antigen-induced arthritis in mice

IL-17 is an important cytokine in the physiopathology of rheumatoid arthritis (RA). However, its participation in the genesis of nociception during RA remains undetermined. In this study, we evaluated the role of IL-17 in the genesis of articular nociception in a model of antigen (mBSA)-induced arthritis. We found that mBSA challenge in the femur-tibial joint of immunized mice induced a dose-and time-dependent mechanical hypernociception. The local IL-17 concentration within the mBSA-injected joints increased significantly over time. Moreover, co-treatment of mBSA challenged mice with an antibody against IL-17 inhibited hypernociception and neutrophil recruitment. In agreement, intraarticular injection of IL-17 induced hypernociception and neutrophil migration, which were reduced by the pre-treatment with fucoidin, a leukocyte adhesion inhibitor. The hypernociceptive effect of IL-17 was also reduced in TNFR1(-/-) mice and by pre-treatment with infliximab (anti-TNF antibody), a CXCR1/2 antagonist or by an IL-1 receptor antagonist. Consistent with these findings, we found that IL-17 injection into joints increased the production of TNF-alpha, IL-1 beta and CXCL1/KC. Treatment with doxycycline (non-specific MMPs inhibitor), bosentan (ET(A)/ET(B) antagonist), indomethacin (COX inhibitor) or guanethidine (sympathetic blocker) inhibited IL-17-induced hypernociception. IL-17 injection also increased PGE(2) production, MMP-9 activity and COX-2, MMP-9 and PPET-1 mRNA expression in synovial membrane. These results suggest that IL-17 is a novel pro-nociceptive cytokine in mBSA-induced arthritis, whose effect depends on both neutrophil migration and various pro-inflammatory mediators, as TNF-alpha, IL-1 beta, CXCR1/2 chemokines ligands, MMPs, endothelins, prostaglandins and sympathetic amines. Therefore, it is reasonable to propose IL-17 targeting therapies to control this important RA symptom. (C) 2009 International Association for the Study of Pain. Published by Elsevier B. V. All rights reserved.

Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model

Aim of the study: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. it comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). Material and methods: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII), and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. Results: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. Conclusions: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

DNA mismatch repair gene methylation in gastric cancer in individuals from northern Brazil

Gastric cancer is one of the most common malignancies. DNA methylation is implicated in DNA mismatch repair genes deficiency. In the present study, we evaluated the methylation status of MLH1, MSH2, MSH6 and PMS2 in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosal of gastric cancer patients from Northern Brazil. We found that none of the nonneoplastic samples showed methylation of any gene promoter and 50% of gastric, cancer samples showed at least one methylated gene promoter. Methylation frequencies of MLH1, MSH2, MSH6 and PMS2 promoter were 21.74%, 17.39%, 0% and 28.26% respectively in gastric cancer samples. MLH1 and PMS2 methylation were associated with neoplastic samples compared to nonneoplastic ones. PMS2:? methylation was associated with diffuse- and intestinal-type cancer compared with normal controls. Intestinal-type cancer showed significant association with MLH1 methylation. Diffuse-type cancer was significantly associated with MSH2 methylation. Our findings show differential gene methylation in tumoral tissue, which allows us to conclude that methylation is associated with gastric carcinogenesis. Methylation of mismatch repair genes was associated with gastric carcinogenesis and may be a helpful tool for diagnosis, prognosis and therapies. However, MSH6 does not seem to be regulated by methylation in our samples.

Mucopolysaccharidoses in Brazil: What happens from birth to biochemical diagnosis?

Mucopolysaccharidoses (MPS) form a group of inherited metabolic disorders characterized by intralysosomal storage of glycosaminoglycans. This study aimed to investigate the path followed by Brazilian patients from birth to diagnosis. An interview was conducted with patient`s parents or guardians with subsequent review of patient`s medical records. One hundred thirteen patients with MPS were included (MPS I: 18, MPS II: 43, MPS III: 2, MPS IIIB: 3, MPS IIIC: 1, MPS IVA: 15, MPS IVB: 1, MPS VI: 29, MPS VII: 1) from 97 families. Median age at the onset of sings/symptoms was 18 months (MPS I: 18, MPS II: 24, MPS IVA: 8, MPS IV: 8). Skeletal abnormalities (for MPS IVA and MPS VI), joint contracures (for MPS II), and typically first signs/symptoms. Several health professionals were involved in patient`s care as of the onset of symptoms until biochemical diagnosis was established. Median age at diagnosis was 76 months (MPS I: 75, MPS II: 95, MPS IVA: 75, MPS VI: 52). Considering the group as a whole, there was a 4.8-year delay between the onset of signs/symptoms and establishment of the diagnosis. Considering that specific therapies are available for some of these disorders and that early treatment is likely to change more favorably the natural history of the disease, efforts should be made to minimize this delay. We believe that this situation can be improved by measures that both increase awareness of health professionals about MPS and improve access to diagnostic tests. (C) 2008 Wiley-Liss, Inc.

Basal levels of DNA damage detected by micronuclei and comet assays in untreated breast cancer patients and healthy women

Breast cancer is the second most frequent type of cancer worldwide and is the most common malignant disease among women. Risk factors for breast cancer include early menarche, late menopause, hormonal therapies, exposure to environmental pollutants, smoking and alcohol use. However, increased or prolonged exposure to estrogen is the most important risk factor. It has been suggested that accumulation of DNA damage may contribute to breast carcinogenesis. Epidemiological studies suggest that cytogenetic biomarkers such as micronuclei in peripheral blood lymphocytes may predict cancer risk because they indicate genomic instability in target tissues. The objective of the present study was to evaluate the frequencies of micronuclei and the extent of DNA damage detected by comet assay in peripheral blood lymphocytes of untreated breast cancer patients and healthy women. The study was conducted using peripheral blood lymphocytes from 45 women diagnosed for Ductal ""in situ"" or invasive breast carcinoma and 85 healthy control women. Micronuclei and comet assays were performed to detect spontaneous DNA damage. The results showed that micronuclei frequencies and tail intensity, detected by comet assay, were significantly higher in the breast cancer group than in controls. The levels of DNA damage were similar in smokers and non-smokers, and aging did not influence the frequencies of micronuclei or tail intensity values observed in either group. In conclusion, the present work demonstrates higher levels of DNA damage in untreated breast cancer patients than in healthy women.

Micro-Positron Emission Tomography in the Evaluation of Trypanosoma cruzi-Induced Heart Disease: Comparison with Other Modalities

Noninvasive assessment of cardiac structure and function is essential to understand the natural course of murine infection with Trypanosoma cruzi. Magnetic resonance imaging (MRI) and echocardiography have been used to monitor anatomy and function; positron emission tomography (PET) is ideal for monitoring metabolic events in the myocardium. Mice infected with T. cruzi (Brazil strain) were imaged 15-100 days post infection (dpi). Quantitative (18)F-FDG microPET imaging, MRI and echocardiography were performed and compared. Tracer ((18)F-FDG) uptake was significantly higher in infected mice at all days of infection, from 15 to 100 dpi. Dilatation of the right ventricular chamber was observed by MRI from 30 to 100 dpi in infected mice. Echocardiography revealed significantly reduced ejection fraction by 60 dpi. Combination of these three complementary imaging modalities makes it possible to noninvasively quantify cardiovascular function, morphology, and metabolism from the earliest days of infection through the chronic phase.

Methotrexate Withdrawal at 6 vs 12 Months in Juvenile Idiopathic Arthritis in Remission A Randomized Clinical Trial

Context Novel therapies have improved the remission rate in chronic inflammatory disorders including juvenile idiopathic arthritis (JIA). Therefore, strategies of tapering therapy and reliable parameters for detecting subclinical inflammation have now become challenging questions. Objectives To analyze whether longer methotrexate treatment during remission of JIA prevents flares after withdrawal of medication and whether specific biomarkers identify patients at risk for flares. Design, Setting, and Patients Prospective, open, multicenter, medication-withdrawal randomized clinical trial including 364 patients (median age, 11.0 years) with JIA recruited in 61 centers from 29 countries between February 2005 and June 2006. Patients were included at first confirmation of clinical remission while continuing medication. At the time of therapy withdrawal, levels of the phagocyte activation marker myeloid-related proteins 8 and 14 heterocomplex (MRP8/14) were determined. Intervention Patients were randomly assigned to continue with methotrexate therapy for either 6 months (group 1 [n = 183]) or 12 months (group 2 [n = 181]) after induction of disease remission. Main Outcome Measures Primary outcome was relapse rate in the 2 treatment groups; secondary outcome was time to relapse. In a prespecified cohort analysis, the prognostic accuracy of MRP8/14 concentrations for the risk of flares was assessed. Results Intention-to-treat analysis of the primary outcome revealed relapse within 24 months after the inclusion into the study in 98 of 183 patients (relapse rate, 56.7%) in group 1 and 94 of 181 (55.6%) in group 2. The odds ratio for group 1 vs group 2 was 1.02 (95% CI, 0.82-1.27; P=.86). The median relapse-free interval after inclusion was 21.0 months in group 1 and 23.0 months in group 2. The hazard ratio for group 1 vs group 2 was 1.07 (95% CI, 0.82-1.41; P=.61). Median follow-up duration after inclusion was 34.2 and 34.3 months in groups 1 and 2, respectively. Levels of MRP8/14 during remission were significantly higher in patients who subsequently developed flares (median, 715 [IQR, 320-1110] ng/mL) compared with patients maintaining stable remission (400 [IQR, 220-800] ng/mL; P=.003). Low MRP8/14 levels indicated a low risk of flares within the next 3 months following the biomarker test (area under the receiver operating characteristic curve, 0.76; 95% CI, 0.62-0.90). Conclusions In patients with JIA in remission, a 12-month vs 6-month withdrawal of methotrexate did not reduce the relapse rate. Higher MRP8/14 concentrations were associated with risk of relapse after discontinuing methotrexate.

Long-term treatment of familial male-limited precocious puberty (testotoxicosis) with cyproterone acetate or ketoconazole

Background Familial male-limited precocious puberty (FMPP) or testotoxicosis is a rare gonadotrophin-independent form of sexual precocity caused by constitutively activating mutations of the LH receptor. Several clinical therapeutic approaches have been reported for this disorder, but with a paucity of long-term outcome data. Objective To evaluate the long-term treatment of testotoxicosis with cyproterone acetate or ketoconazole. Design A multicentric retrospective clinical study. Patients Ten boys from eight unrelated Brazilian families who carried known LH-receptor activating mutations were treated with 70 mg/m(2) cyproterone acetate (n = 5) or 10 mg/kg ketoconazole (n = 5) for a mean period of 5 and 8 years, respectively. Measurements Chronological and bone ages, bone age/chronological age ratio, target height (TH) range, adult height, basal and GnRH-stimulated gonadotrophin levels and basal testosterone levels were assessed. Results Growth velocity decreased significantly during treatment with cyproterone acetate or ketoconazole when compared to pretreatment value in each group (P < 0.05). Bone age/chronological age ratio decreased significantly after cyproterone acetate or ketoconazole therapy. Basal testosterone levels were significantly lower in patients undergoing ketoconazole compared to cyproterone acetate treatment [0.6 +/- 0.3 nmol/l (42 +/- 21 ng/dl) vs. 5.6 +/- 4.0 nmol/l (392 +/- 280 ng/dl); P < 0.05], as expected. Secondary gonadotrophin-dependent precocious puberty occurred at a similar frequency (40%) in both groups. Five patients have attained adult height and two patients have already reached 90% of their adult height. Two of them achieved their TH range and one patient, for whom TH was not available, had an adult height of 0.3 SDS. Four boys (two in each group) did not attain their TH range. Conclusion Long-term treatment with cyproterone acetate or ketoconazole resulted in similar outcomes without important side-effects in boys with testotoxicosis. However, both therapies showed limited efficacy in attaining normal adult height.

Risk stratification in neonates and infants submitted to cardiac surgery with cardiopulmonary bypass: A multimarker approach combining inflammatory mediators, N-terminal pro-B-type natriuretic peptide and troponin I

Low cardiac output syndrome (LCOS) is a common problem following cardiac surgery with cardiopulmonary bypass (CPB) in neonates and infants, and its early recognition remains a challenging task. We aimed to test whether a multimarker approach combining inflammatory and cardiac markers provides complementary information for prediction of LCOS and death in children submitted to cardiac surgery with CPB. Forty-six children younger than 18 months with congenital heart defects were prospectively enrolled. No intervention was made. Blood samples were collected pre-operatively, during CPB and post-operatively (PO) for measurement of interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, cardiac troponin I (cTnI) and N-terminal pro-B-type natriuretic peptide (NT-proBNP). Clinical data and outcome variables were recorded. Logistic regression was used to identify predictors of LCOS and death. Multivariate logistic regression identified pre-operative NT-proBNP and IL-8 4 h PO as independent predictors of LCOS, while cTnI 4 h PO and CPB length were independent predictors of death. The use of inflammatory and cardiac markers in combination improved sensitivity, negative predictive value and accuracy of the models. In conclusion, the combined assessment of inflammatory and cardiac biochemical markers can be useful for identifying young children at increased risk for LCOS and death after heart surgery with CPB. (C) 2008 Elsevier Ltd. All rights reserved.

Advances in Imaging of Osteoarthritis and Cartilage

Osteoarthritis (OA) is the most frequent form of arthritis, with major implications for individual and public health care without effective treatment available. The field of joint imaging, and particularly magnetic resonance (MR) imaging, has evolved rapidly owing to technical advances and the application of these to the field of clinical research. Cartilage imaging certainly is at the forefront of these developments. In this review, the different aspects of OA imaging and cartilage assessment, with an emphasis on recent advances, will be presented. The current role of radiography, including advances in the technology for joint space width assessment, will be discussed. The development of various MR imaging techniques capable of facilitating assessment of cartilage morphology and the methods for evaluating the biochemical composition of cartilage will be presented. Advances in quantitative morphologic cartilage assessment and semiquantitative whole-organ assessment will be reviewed. Although MR imaging is the most important modality in imaging of OA and cartilage, others such as ultrasonography play a complementary role that will be discussed briefly.

Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D(2)) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for `in-situ` hybridisation study using (35)S-labeled oligonucleotide probe complementary to D(2) receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D(2) receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D(2) receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: Evaluation of reduced glutathione levels and matrix metalloproteinase secretion

Background and purpose: Calendula officinalis flowers have long been employed time in folk therapy, and more than 35 properties have been attributed to decoctions and tinctures from the flowers. The main uses are as remedies for burns (including sunburns), bruises and cutaneous and internal inflammatory diseases of several origins. The recommended doses are a function both of the type and severity of the condition to be treated and the individual condition of each patient. Therefore, the present study investigated the potential use of Calendula officinalis extract to prevent UV irradiation-induced oxidative stress in skin. Methods: Firstly, the physico-chemical composition of marigold extract(ME) (hydroalcoholic extract)was assessed and the in vitro antioxidant efficacy was determined using different methodologies. Secondly, the cytotoxicity was evaluated in L929 and HepG2 cells with the MTT assay. Finally, the in vivo protective effect of ME against UVB-induced oxidative stress in the skin of hairless mice was evaluated by determining reduced glutathione (GSH) levels and monitoring the secretion/activity of metalloproteinases. Results and conclusions: The polyphenol, flavonoid, rutin and narcissin contents found in ME were 28.6 mg/g, 18.8 mg/g, 1.6 mg/g and 12.2 mg/g, respectively and evaluation of the in vitro antioxidant activity demonstrated a dose-dependent effect of ME against different radicals. Cytoxicity experiments demonstrated that ME was not cytotoxic for L929 and HepG2 cells at concentrations less than or equal to of 15 mg/mL However, concentrations greater than or equal to 30 mg/mL, toxic effects were observed. Finally, oral treatment of hairless mice with 150 and 300 mg/kg of ME maintained GSH levels close to non-irradiated control mice. In addition, this extract affects the activity/secretion of matrix metalloproteinases 2 and 9 (MMP-2 and -9) stimulated by exposure to UVB irradiation. However, additional studies are required to have a complete understanding of the protective effects of ME for skin. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Effect of Acute and Chronic Physical Exercise on Patients with Periodic Leg Movements

ESTEVES, A. M., M. T. DE MELLO, M. PRADELLA-HALLINAN, and S. TUFIK. Effect of Acute and Chronic Physical Exercise on Patients with Periodic Leg Movements. Med. Sci. Sports Exerc., Vol. 41, No. 1,. pp. 237-242, 2009. Purpose: Nonpharmacological interventions may lead to an improvement in sleep quality. The objective of our study was to evaluate the effects of acute intensive exercise and chronic exercise on sleep patterns in patients with periodic leg movements (PLM). Methods: The study involved acute and chronic exercise. The acute intensive exercise group consisted of 22 volunteers who underwent a maximum effort test and a polysomnography (PSG) on the same night. The chronic exercise group included. 11 patients who performed 72 physical training sessions undergoing three PSG studies on the night of sessions 1, 36, and 72. Blood samples were collected from both acute and chronic groups for beta-endorphin dosage. Results: Our results showed that both forms of physical exercise lowered PLM levels. The acute physical exercise increased sleep efficiency, rapid eye movement (REM) sleep, and reduced wake after sleep onset, whereas the chronic physical exercise increased sleep efficiency, REM sleep, and reduced sleep latency. We also found a significant negative correlation between beta-endorphin release after acute intensive exercise and PLM levels (r = -0.63). Conclusion: Physical exercise may improve sleep patterns and reduce PLM levels. The correlation between beta-endorphin release after acute intensive exercise and PLM levels might be associated with the impact physical exercise has on the opiodergic system. We suggest that physical exercise may be a useful nonpharmacological treatment for PLM.

Imaging in osteoarthritis: what have we learned and where are we going?

Osteoarthritis (OA) is a widely prevalent disease of the whole joint including cartilage, bone and soft tissues. Increasing importance of imaging including assessment of all joint structures has been recognized recently. Conventional radiography is still the first and most commonly used imaging technique for evaluation of a patient with a known or suspected diagnosis of OA. However, limitations have been revealed by recent MRI-based knee OA studies. MRI plays a crucial role in understanding the natural history of the disease and in guiding future therapies due to its ability to image the knee as a whole organ and to directly and three-dimensionally assess cartilage morphology and composition. It is crucial to use the appropriate MR pulse sequences to assess various OA features, and thus support from experienced musculoskeletal radiologists should be sought for study design, image acquisition and interpretation. The aim of this article is to describe the roles and limitations of conventional radiography and MRI in imaging of OA, and also to give insight into the use of other modalities such as ultrasound, scintigraphy, computed tomography (CT) and CT arthrography in clinical practice and research in OA, particularly focusing on the assessment of knee OA in the tibiofemoral joint.

Characterization of Equine Adipose Tissue-Derived Progenitor Cells Before and After Cryopreservation

In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.